A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre–lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.


Figure S1
. Map of the plasmid pMT85Pgenta-PSlacZ-pRS313.This plasmid derives from the transposon based-plasmid pMT85PStet(M)-PSlacZ-pRS313 1 .It harbors the aacA-aphD gentamicin resistance gene (gmR) and a transposase encoding gene (tnp) flanked by two inverted repeats (IR) from the Tn4001 transposon.This plasmid also contains (i) the ColE1 origin and an ampicillin resistance gene (ampR) for propagation and selection in Escherichia coli, (ii) an autonomous replicating sequence (ARSH4), a centromere (CEN6) and a histidine marker (HIS3) for propagation and selection in the yeast Saccharomyces cerevisiae and (iii) the betagalactosidase encoding gene (lacZ) under the spiralin promoter (PS) that can be used to screen some recombinant mycoplasma species.Recombination templates required for the CReasPy-cloning method were amplified from the pMT85Pgenta-PSlacZ-pRS313  S1).The plasmid map was created using SnapGene.

Figure S2
: Schematic representation of the recombination templates used during the CReasPy-cloning experiment.Two recombination templates were used in this study.Both carry the yeast elements (ARSH4, CEN6, HIS3), the gentamicin resistance marker (gmR) and, at each extremity, 60pb-hooks with sequences identical to those flanking the target gene (light grey boxes).The recombination template at the bottom also includes two loxP sites (lox66 and lox71) 2 so that DNA sequences added during the genome engineering cycle can be removed if required.The size of the recombination templates is indicated.More specifically, the integration status of pMccpO2 plasmids was studied by PCR using 3 pairs of primers after 3 and 6 passages in either selective or non-selective broth.One passage corresponds to the transfer of 10 μL of mycoplasma culture to 1 mL of medium (1/100 dilution) followed by an incubation period of ≥24h at 37 °C.A total of 9 clones from 3 independent transformation experiments (3 per experiment) were tested and the primer pairs used for the analysis were as follows: - To be more precise, tetracycline-resistant colonies were picked in 1mL of m-Hayflick medium containing 0.5µg/µL tetracycline and subcultured for a total of 6 passages.The first 3 passages were performed in m-Hayflick medium containing 0.5µg/µL tetracycline (part A, red column) while passages 4 to 6 were carried out either in the presence (part A, blue column) or absence (part A, green column) of tetracycline.Then, to monitor the evolution of these clones and plasmid stability over time, 3 additional passages were performed in m-Hayflick medium either with tetracycline (part A, blue column) or without tetracycline (part A, green column).
When the selection pressure was maintained in the culture medium for a further 3 passages (part A, blue column), the PCR profile changed for some clones and not for others compared to 3P:  Of the four clones carrying free plasmids at 3P, three (cl.6.1, 6.3 and 10.1, blue box) maintained an unchanged PCR profile at 6P, meaning that they still carried the plasmid as a free molecule in the cytoplasm after 6 in vitro passages and one (cl 2.1) showed a different PCR profile.In this clone, a PCR amplicon was detected with both primer pairs, indicating that the plasmid was free in some cells and integrated at the oriC in others. Of the five clones showing a mixed profile at 3P, all again showed a two-band profile at 6P (indicating a mixed population).In three of them (cl2.4,6.4 and 10.4), the band at 2,393 bp (free plasmid) was barely visible, suggesting that in these clones the majority of the cells had integrated the plasmid at the oriC.In summary, after 6 passages in liquid medium plus tetracycline, the plasmid was found totally free in 3 out of 9 clones and in mixture in the other 6.This experiment showed that when selection pressure is maintained over time, the pMccpO2 plasmid tends to integrate the genome, first generating mixed clones and then pure clones consisting solely of cells in which the plasmid sequence is integrated at the origin of replication.
Interestingly, when the selection pressure was removed from the culture medium from passage 3 onwards (part A, green column), the PCR profile changed for several clones, but not in the same way:  Clones carrying plasmids in free form at 3P (cl.2.1, 6.1, 6.3 and 10.1) lost them all after "tetracyclinefree passages", as shown both by the absence of amplicon at 4,030bp with the primer pair PIVT-TETF1/ PIVT-TETR and the presence of amplicon at 2,393bp with the primer pair Mccp-rpnA-F/Mccp-dnaN-R.  Finally, among, the 5 clones with a mixed profile at 3P, 4 showed an unchanged profile at 6P (cl.2.2, 2.4, 6.4, 10.4) and one (cl10.3)appeared to be made up of cells that had lost the plasmid.This suggests that cells with an intact origin of replication and no plasmid to replicate had a growth advantage over the others.In summary, after 6 passages in liquid medium without antibiotics, pMccpO2 plasmid was found free in 5 out of 9 clones (blue box), and in mixture in the other 4.
These data confirmed that the pMccO2 plasmid is a replicative plasmid, that it can be maintained free in the form of extra-chromosomal molecules in certain clones up to at least 6 passages after transformation and that it can be lost in an antibiotic-free environment.We concluded from these experiments that we could use the pMccpO2 plasmid to express the heterologous cre gene over a few generations, and that it would then be possible to get rid of the pMccpO2-CRE plasmid simply by removing the selection pressure.0 --* Transformed cells were seeded on m-Hayflick selective plates directly after transformation ** Transformation efficiencies were not calculated for assays performed with plasmids carrying a puromycin resistance cassette, because genotypic analyses showed that the colonies that appeared on the plates containing 8 µg.mL-1 of puromycin were all spontaneous mutants.***Mccp transformation experiment with plasmid pMT85PStet(M)-PSlacZ-PRS313 was carried out in an independent experiment.During that experiment, the transformation efficiency with pIVT-1 (used as a positive control) was equal to 1.3 x 10 -9 transformants CFU/total CFU/µg of plasmids and that of pMT85PStet(M)-PSlacZ-PRS313 was equal to 1.85 x 10 -10 transformants CFU/total CFU/µg of plasmids, i.e. around 10 times lower.**** Plasmid used as positive control.
Figure S1.Map of the plasmid pMT85Pgenta-PSlacZ-pRS313.This plasmid derives from the transposon based-plasmid pMT85PStet(M)-PSlacZ-pRS313 1 .It harbors the aacA-aphD gentamicin resistance gene (gmR) and a transposase encoding gene (tnp) flanked by two inverted repeats (IR) from the Tn4001 transposon.This plasmid also contains (i) the ColE1 origin and an ampicillin resistance gene (ampR) for propagation and selection in Escherichia coli, (ii) an autonomous replicating sequence (ARSH4), a centromere (CEN6) and a histidine marker (HIS3) for propagation and selection in the yeast Saccharomyces cerevisiae and (iii) the betagalactosidase encoding gene (lacZ) under the spiralin promoter (PS) that can be used to screen some recombinant mycoplasma species.Recombination templates required for the CReasPy-cloning method were amplified from the pMT85Pgenta-PSlacZ-pRS313 plasmid using the primer pairs MccpPeptS41pMT85gentPRS-F / MccpPeptS41pMT85gentPRS-F (4,481bp) and primer pairs MccpPeptS41LOX66pMT85gentPRS-F / MccpPeptS41LOX71pMT85gentPRS-F (4,549bp).Primers were composed of 20-bases identical to the plasmid sequence and ~60-bases floating tails identical to the sequences surrounding the Mccp target gene MCCP001_RS01320.Primers pair MccpPeptS41LOX66pMT85gentPRS-F / MccpPeptS41LOX71pMT85gentPRS-F also includes a 34-base-long lox site (TableS1).The plasmid map was created using SnapGene.

Figure S3 .
Figure S3.Maps of the OriC replicative plasmids pMccpO1 and pMccpO2.These plasmids derived from the pBluescript II KS (-) commercial plasmid (2,961bp), which harbors the ColE1 origin and an ampicillin resistance gene (ampR) for propagation and selection in Escherichia coli.Both plasmids also contain a 1,974bp BamHI DNA cassette harboring the Mccp dnaA gene surrounded by its intergenic regions for replication in Mccp cells, as well as a 4,034bp PstI DNA cassette originating from the pIVT-1 with a tetracycline resistance gene (tet(M)) for mycoplasma selection.PMccpO1 differs from pMccpO2 by the orientation of the BamHI DNA cassette.Plasmid maps were created using SnapGene.

Figure S4 .
Figure S4.Map of the OriC replicative plasmid pMccpO2-CRE.This plasmid derived from plasmid pMccpO2, described in Figure S3.It harbors the cre gene under the gentamicin promoter.The plasmid map was created using SnapGene.

Figure S5 :
Figure S5: Full images used to produce the panels in Figure 2. The sections spliced together are highlighted with blue rectangles.

FigureFigure S7 .
Figure S7.See legend on next page PIVT-TETF1/ PIVT-TETR : this primer pair (specific of the tet(M) resistance cassette) enables specific amplification of a 4,030 bp DNA fragment in Mccp cells carrying either extrachromosomal or genomeintegrated pMccpO2 molecules (part B, top and bottom panels).-Mccp-rpnA-F/Mccp-dnaN-R : this primer pair enables specific amplification of a 2,393-bp DNA fragment encompassing the Mccp dnaA gene in Mccp WT cells and in Mccp cells carrying extrachromosomal pMccpO2 molecules (part B, Top panel).-Mccp-rpnA-F/AmpR-R : this primer pair enables specific amplification of a 3,155-bp DNA fragment in Mccp cells that have integrated the pMccpO2 plasmid at the chromosomal dnaA gene by homologous recombination via simple crossing over (part B, Bottom panel).

After 3
passages in m-Hayflick plus tetracycline (part A, red column), all clones showed a 4,030bp-tet(M) amplicon (gel at the top), indicating that they all possessed the pMccpO2 plasmid.Then, PCR performed with primer pairs Mccp-rpnA-F/Mccp-dnaN-R and Mccp-rpnA-F/AmpR-R (which discriminate between clones presenting the plasmid either free or integrated into the chromosome) revealed 2 types of PCR profile:  The first profile (cl.2.1, 6.1, 6.3 and 10.1, blue box) showed the presence of an amplicon only with the primer pair Mccp-rpnA-F/Mccp-dnaN-R indicating that the plasmid is free in these clones. The second profile (cl.2.2, 2.4, 6.4, 10.3, 10.4) showed the presence of an amplicon with both pairs of primers, suggesting the occurrence of a mixed population of cells, carrying either free or integrated plasmids (or both).Therefore, after 3 passages in liquid medium with antibiotics, the plasmid was found totally free in 4 of the 9 clones analyzed and in mixture in 5 others.The finding of extrachromosomal DNA molecules in Mccp transformants here clearly demonstrates that the oriC region predicted in silico for Mccp enables the replication of plasmids and that pMccpO2 is a replicative plasmid.

Figure S9 :
Figure S9: Full images used to produce the panels in Figure 4.The sections spliced together are highlighted with blue rectangles.

Table S1 :
Primers and Plasmids (see Excel document)